Many fermentation organisms convert carbohydrates to ethanol. The most widely used fermentation organisms, brewer's yeast and baker's yeast, are strains of Saccharomyces cerevisiae. Ethanol has significant economic value as beverages, transportation fuels and precursors for other organic compounds.
Fermentation organisms can directly convert glucose, fructose, maltose (glucose dimer) and sucrose (glucose-fructose dimer) to ethanol. Herein, monomers and dimers of glucose and fructose will be referred to as simple sugars and fermentation organisms that convert simple sugars to ethanol will be referred to as yeasts.
Yeasts ferment simple sugars to ethanol in an anaerobic (without oxygen) environment. One mole of glucose or fructose (or 0.5 mole of sucrose) is fermented to 2 moles of ethanol and 2 moles of carbon dioxide and gives off 118 kJ of heat. This means that fermenting an 18% sugar solution will result in a temperature rise of 34° C., which means that cooling of the fermentation medium is required. Fermenting 1 liter of an 18% sugar solution (1 mole of glucose) will also produce 2 moles of carbon dioxide, which has a volume of about 48 liters at 20° C. and atmospheric pressure. A typical yeast ferments most efficiently between 20-40° C. but has significant fermentation activity down to 5° C. (white wine is fermented between 7-15° C.). Yeast cells die gradually at temperatures above 42° C. Saccharomyces cerevisiae is relatively insensitive to pH and will ferment in a pH range from 2.9 to 7.2. This is described in more detail in Arroyo-López, “Effects of temperature, pH and sugar concentration on the growth parameters of Saccharomyces cerevisiae, S. kudriavzevii and their interspecific hybrid,” International journal of food microbiology 131.2 (2009): 120-127, which is hereby incorporated by reference herein.
Most Saccharomyces cerevisiae strains have a diameter of approximately 10 microns. A Saccharomyces cerevisiae strain with a cell size of approximately 5 microns is Thermosacc® Dry, available from Lallemand Biofuels & Distilled Spirits, Duluth, Ga., USA. It produces ethanol concentrations up to 20% by volume (16% by weight), so carbohydrate-rich crops with up to 32% carbohydrates by weight can be fermented by this yeast. This means that a crop can be dehydrated before fermenting so that the resulting ethanol concentration is higher.
Yeast cells adhere to surfaces (such as parenchyma cells) in the presence of simple sugars. This is described Verstrepen and Klis, “Flocculation, adhesion and biofilm formation in yeasts,” Molecular microbiology 60.1 (2006): 5-15, which is hereby incorporated by reference herein.
Saccharomyces cerevisiae is sold in freeze-dried form and is easy to handle. It is classified as GRAS (Generally Recognized as Safe) and is commonly consumed in the average diet—for example, bread is made with Saccharomyces cerevisiae yeast.
Starch is a polymer of glucose and inulin is a polymer of mostly fructose with glucose at one end. Before starch and inulin can be converted by yeast to ethanol, they must first be converted to simple sugars by amylases and inulinases, respectively, or by acids. Starch is insoluble in water in the temperature range for which yeast is active, and only about 5% of inulin is soluble in this same temperature range.
There are amylases available that convert starch to glucose efficiently in the temperature range that yeast operates efficiently. One example is the STARGEN® 002 enzyme formulation from DuPont Industrial Biosciences, USA. This contains an Aspergillus kawachi alpha-amylase expressed in Trichoderma reesei and a gluco-amylase from Trichoderma reesei that work synergistically to hydrolyze granular starch substrate to glucose. The endo-activity, alpha-amylase and exo-activity, gluco-amylase catalyze the complete hydrolysis of granular starch under a variety of ethanol fermentation conditions.
There are inulinases available that convert inulin to fructose efficiently in the temperature range that yeast operates efficiently. One example is the Fructozyme L enzyme formulation available from Novozymes A/S, Denmark.
Many crops contain carbohydrates within storage parenchyma cells. These carbohydrate-rich parenchyma cells generally have 10% to 20% carbohydrates in a single large vacuole with 80% to 90% water. These carbohydrates generally comprise simple sugars and polysaccharides. Herein, the parts of these carbohydrate-rich crops that contain a significant mass of carbohydrate-rich parenchyma cells will be referred to as carbohydrate-rich parenchyma tissue. All crops with carbohydrate-rich parenchyma tissue contain some amount of simple sugars in the parenchyma cells and some contain a significant amount of polysaccharides.
There are two types of crops with carbohydrate-rich parenchyma tissue, monocotyledons (monocots) in the grass family (Poaceae and Dioscorea) and dicotyledons (dicots). They differ in the way the parenchyma cells adhere to each other. Monocots adhere through both pectin and hemicellulose in the middle lamella and dicots adhere through pectin in the middle lamella.
The most widely cultivated crops with carbohydrate-rich parenchyma tissue in the stalks are sugar cane (Saccharum officinarum), sweet sorghum (Sorghum bicolor) and tropical maize hybrid (Zea mays). These are all monocots in the grass family (Poaceae). Sugar cane and tropical maize hybrid contain simple sugars in the storage parenchyma cells and sweet sorghum contains 90% simple sugars and 10% starch in the storage parenchyma cells.
The most widely cultivated crops with carbohydrate-rich parenchyma tissue in tubers are potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), yam (genus Dioscorea) and Jerusalem artichoke (Helianthus tuberosus). Potato, sweet potato, cassava and Jerusalem artichoke are dicots while yam is a monocot. Potato, sweet potato, cassava and yam contain starch in the storage parenchyma cells and Jerusalem artichoke contains inulin in the storage parenchyma cells.
The most widely cultivated crops with carbohydrate-rich parenchyma tissue in fruits are apples, grapes and oranges. These are all dicots and contain glucose and fructose in the storage parenchyma cells.
There are well-known techniques for fermenting crops with carbohydrate-rich parenchyma tissue. Stalks are generally crushed between a series of rollers to extract the juice by bursting the parenchyma cells, and then the juice is separated from residual solids and fermented. Sugar beets are generally cut into small slices approximately 4 mm thick (cossettes) and the sugar is extracted with flowing hot water and is then fermented. Fruits are generally squeezed to extract sugar-rich juice which is then fermented. Starchy crops are generally converted to ethanol by heating the tuber above the gelatinization temperature and using amylases with the gelatinized starch, followed by fermentation of the glucose. Inulin-rich crops are generally fermented by heating until the inulin solubilizes, extracting the juice, using acid hydrolysis to convert to fructose and then fermenting the fructose. All of these techniques are quite capital-intensive.
The storage parenchyma cells in carbohydrate-rich parenchyma tissue are thin-walled polyhedral cells. Sugar beet parenchyma cells have a diameter of approximately 100 microns with a wall thickness of about 2 microns. The parenchyma cells in stalks are approximately 360 microns long and 60 microns in diameter with a wall thickness of about 2 microns. The characteristics of storage parenchyma tissue are described in more detail in Gibson, “The hierarchical structure and mechanics of plant materials,” Journal of The Royal Society Interface 9.76 (2012): 2749-2766, which is hereby incorporated by reference herein.
The parenchyma cells are packed tightly together, but there are small gaps between them because the packing is imperfect. These gaps are known as the apoplast, or intercellular space. These gaps are interconnected, and water can flow through the parenchyma tissue through these gaps. There is more detail about water flow through the apoplast in Steudle, “Water transport in plants: role of the apoplast,” Plant and Soil 187.1 (1996): 67-79, which is hereby incorporated by reference herein.
Water flows through the apoplast in sugar beet parenchyma tissue in the axial direction (up/down) but is limited in the radial direction (in/out) by the casparian strips in the roots. This is described in more detail in Amodeo, “Radial and axial water transport in the sugar beet storage root,” Journal of Experimental Botany 50.333 (1999): 509-516, which is hereby incorporated by reference herein.
Similarly, water flows through the apoplast in parenchyma tissue of carbohydrate-rich stalks in the axial direction, but is limited by the internode length (the continuous sections of the stalk). Water doesn't flow in the radial direction because the outer part of the stalk is impenetrable to water. The internode of most carbohydrate-rich stalks is between 100 mm and 300 mm in length.
The apoplast (intercellular space) of sugar cane is sufficiently large to be colonized by a variety of bacteria. This is described in more detail in Dong, “A nitrogen-fixing endophyte of sugarcane stems (a new role for the apoplast),” Plant Physiology 105.4 (1994): 1139-1147 and in Tejera, “Nitrogen compounds in the apoplastic sap of sugarcane stem: Some implications in the association with endophytes,” Journal of plant physiology 163.1 (2006): 80-85, both of which are hereby incorporated by reference herein. Similarly, the apoplast of other species of carbohydrate-rich parenchyma tissue is large enough to be colonized by bacteria.
It is possible to fill the apoplast of carbohydrate-rich parenchyma tissue by using vacuum infusion (also called vacuum impregnation). This involves surrounding the parenchyma tissue with a liquid, applying a vacuum, waiting for liquid and gas to be expelled from the parenchyma tissue, releasing the vacuum and waiting for the liquid to fill the apoplast. This is described in more detail in Gras, “The response of some vegetables to vacuum impregnation,” Innovative Food Science & Emerging Technologies 3.3 (2002): 263-269, which is hereby incorporated by reference herein.
Carbohydrate-rich parenchyma tissue often contains up to 20% (by mass) carbohydrates. The parenchyma cell wall provides strength to the parenchyma cell and the cell membrane keeps the contents of the cell from leaking out of the cell. The cell wall is permeable to sucrose and other simple sugars. The cell membrane can be denatured by heat, generally above 70° C., which increases the diffusion coefficient of simple sugars through the cell membrane. This is the technique normally used to extract sucrose from sugar beets—the cell membrane is denatured by heat and then the sucrose diffuses out of the sugar beet into hot water. The diffusion coefficient of sucrose through denatured sugar beet tissue is about five times higher than through non-denatured sugar beet tissue, which is described in more detail in Bessadok-Jemai et al., “Modeling the kinetic of solute diffusion from sugarbeet particles based on electric conductivity measurements,” International Journal of Physical Sciences 6.28 (2011): 6464-6468, which is hereby incorporated by reference herein.
Parenchyma cells can be macerated (separated from each) by either heat or enzymes. When the parenchyma cells are macerated, the cell membrane is also breached, both from mechanical action and from enzymes that are released from the cell wall. This causes the contents of the vacuoles to leak out of the parenchyma cells and causes enzymes to more easily diffuse into the vacuoles. This also provides a retting action, where the water in the parenchyma cells can be more easily removed by squeezing or evaporation. Any combination of pectin lyase, pectate lyase and polygalacturanase macerate parenchyma cells in dicots, while pectin lyase and xylanase macerate parenchyma cells in monocots. This is described in Ishii, “Enzymes for the isolation of protoplasts,” Plant Protoplasts and Genetic Engineering I. Springer Berlin Heidelberg, 1989, 23-33, which is hereby incorporated by reference herein.
Pectate lyase and polygalacturanase, when they degrade pectin, also produce methanol which is often an undesirable product when producing ethanol. Pectin lyase degrades pectin without producing methanol as a byproduct and xylanase doesn't produce any alcohols. There are pectin lyases available that operate in the same pH and temperature range as yeast, in particular pectin lyase from Aspergillus niger, with an optimum pH of 5.5 and an optimum temperature of 35° C. This is described in Yadav et al., “Pectin lyase: a review,” Process Biochemistry 44.1 (2009): 1-10, which is hereby incorporated by reference herein. One example of a pectin lyase that operates in the same pH and temperature range of yeast is the “Pectinei® Ultra Color” enzyme formulation available from Novozymes A/S, Denmark.
When fermenting, yeast produces large amounts of carbon dioxide (CO2). Carbonic acid is formed by the dissolution of CO2 in water. When fermenting, the partial pressure of CO2 is 100 kPa (1 atm) and the pH of this solution is about 3.92. Yeast ferments well at this pH, pectin lyase enzymes from Aspergillus niger (such as Pectinex Ultra Color) have significant activity at this pH, granular starch hydrolyzing enzymes (such as STARGEN) have significant activity at this pH, and inulinase enzymes (such as Fructozyme L) have significant activity at this pH. Similarly, all of these enzymes have significant activity in the temperature range of yeast (25° C. to 40° C.).
The harvest temperature of sugar beet can be quite cold, often below 10° C. and the harvest temperature of sugar cane, sweet sorghum and tropical maize hybrids can be below 20° C. However, the heat released by fermentation of simple sugars in the apoplast of carbohydrate-rich parenchyma tissue will rapidly increase the temperature of this tissue to the temperature range where enzymes have significant activity.
The rate of fermentation is heavily influenced by the concentration of yeast cells. Sugar cane fermentation in typical mills in Brazil can take between 6 to 10 hours, but this requires high concentrations (10% w/w) of yeast and yeast cell recycling. This is described in more detail in Basso, “Ethanol production in Brazil: the industrial process and its impact on yeast fermentation,” INTECH Open Access Publisher, 2011, which is hereby incorporated by reference herein. Wine or beer fermentation, with lower concentrations of yeast, can take up to a week.
One significant problem with current techniques for fermenting sugars to ethanol is bacterial contamination, in particular contamination by Lactobacillus. Without wishing to be bound by any particular theory, it is believed that turbulent mixing propagates bacteria throughout the fermentation medium, and since the contaminating bacteria can out-compete yeast, there is significant contamination. Without mixing, and without a gradient in sugar concentration (caused by uniformly distributing yeast in the carbohydrate-rich parenchyma tissue), any possible bacterial contamination remains localized and is unable to outcompete yeast across the whole biomass volume. This is described in Kundiyana et al., “Influence of temperature, pH and yeast on in-field production of ethanol from unsterilized sweet sorghum juice,” biomass and bioenergy 34.10 (2010): 1481-1486, which is hereby incorporated by reference herein.
Because the parenchyma cells are so small, it takes a lot of energy to crush them or to extract the sugar from them with hot water. Almost 35% of the capital and operating costs of producing sugar from stalks is due to the cost of crushing. Similarly, much of the cost of producing sugar from sugar beets is due to the cost of hot water extraction. The economics of crushing sugar cane is described in more detail in Gbaboa, “Comparative study on cane cutter/juice expeller and roller model Sugarcane juice extraction systems,” INT J CURR SCI 2013, 7: E 55-60, which is hereby incorporated by reference herein. It would reduce the costs of extracting sugars if the need for crushing or hot water extraction could be eliminated.
The bulk density of sugar beets is about 769 kg/m3 and the bulk density of billets (cut sections) of sugar cane, sweet sorghum and tropical maize hybrids (i.e. stalks) is about 350 kg/m3. If the sugar content is about 18%, this results in a sugar density of 138 kg sugar per cubic meter of sugar beets and 63 kg sugar per cubic meter of sugar cane, sweet sorghum and tropical maize hybrids. Since transportation costs are primarily a function of volume (and not weight), and since crops are often harvested significant distances from where they're processed, it is quite expensive to transport sugars at such low densities since only 5% to 10% of the volume of a truck is taken up by sugar. It is desired to reduce the cost of making ethanol from carbohydrate-rich crops by making ethanol at (or close to) the harvest site of these crops, reducing transportation costs.
Parenchyma cells in carbohydrate-rich parenchyma tissue are living tissue and therefore respire (breathe) after harvest. Respiration involves converting oxygen and sugar in the parenchyma cells to carbon dioxide and energy to maintain the cell. After sugar beets are harvested, about 200 g of sugar per day per metric ton of sugar beet are consumed by respiration, and in the first 5 days after harvest about 600 to 1500 g of sugar per day per metric ton of sugar beet are consumed by respiration. If sugar beets are about 18% sugar by weight, there is about 180 kg sugar in a metric ton of sugar beet, resulting in a loss of between 0.3% to 0.8% of sugar per day in the first 5 days and 0.1% of sugar per day in subsequent days. Given that sugar beets can be stored for 100 days before being processed, they can lose up 10% of their sugar content due to respiration. Sugar cane, sweet sorghum and tropical maize lose similar amounts of sugar when being stored. There is a need in the art to reduce the sugar lost to respiration by more rapidly converting carbohydrates to ethanol than current methods. Once the carbohydrates in crops are converted to ethanol, they can be stored for long periods, allowing continuous removal of the ethanol year round. It is desired to more efficiently use the capital invested in roller extraction, ethanol stripping and distillation by using this equipment year round, not just during the harvest season.
If sugar beets are stored in anaerobic (without oxygen) conditions, microorganisms will colonize the beets and after 21 days will completely ferment all sugar in the beets, mostly to lactic acid and acetic acid. Since the outer layer of the sugar beets are often abraded and damaged by harvesting, microorganisms can more easily penetrate the outer layers of the sugar beet, leading to sugar losses due to fermentation to lactic acid and acetic acid. Similarly, sugar cane, sweet sorghum and tropical maize hybrid are more susceptible to microorganisms penetrating the pith because the cane has been cut open into billets during harvesting.
Much of the capital cost and operating cost of producing ethanol from carbohydrate-rich crops is the cost of heating the feedstock. These costs could be reduced (or eliminated) by using the self-heating from energy released by fermentation.
Some techniques for producing ethanol from carbohydrate-rich crops require pretreatment or fermentation inside a pressure vessel. Because pressure vessels have a danger of exploding and require more strength than an unpressurized vessel, it would be beneficial to not require a pressure vessel.
Another significant capital cost and operating cost of producing ethanol from carbohydrate-rich crops is the cost of cooling the fermentation reactor. It would be desirable to use low-cost passive cooling such as blowing air over metal walled tanks or circulating cool carbon dioxide gas through the crop.